The hippocampus in intact animals, in slice preparation, and as isolated neurons in culture offers an experimental opportunity to study both long- term depression (LTD) and long-term potentiation (LTD), forms of synaptic plasticity utilized in learning and memory. Ca2+ is a key signaling molecular regulating synaptic plasticity in the hippocampus and we will focus on several aspects of Ca2+ action including the activation of protein kinases/phosphatases, nitric oxide synthase, and transcription of genes that underlie changes in synaptic function during LTD and LTP. Nitric oxide (no) is a retrograde messenger that we have found to stimulate synaptic release in a Ca2+-independent manner. We have collaborated with Dr. Richard Scheller to demonstrate NO alters protein-protein interactions among the synaptic proteins VAMP, syntaxin, n-secl, and SNAP-25 that may be responsible for such release. We will define, quantitate, and identify the sites of these changes. We will determine which neurotransmitter classes are affected and whether NO alters stimulated release. We have demonstrated a mechanism by which multifunctional CaM kinase II may be switched to a Ca2+ -independent species in a stimulus frequency- dependent manner. In collaboration with Dr. Richard Tsien we will correlate activation of the kinase at various frequencies that elicit LTD or LTP in hippocampal cultures. Immunocytochemistry with phosphoselective Ab and biochemical analysis will compare antagonism between CaM kinase II and calcineurin, a Ca2+-dependent phosphatase. We will examine how Ca2+ can change the sign of the synaptic strength by favoring activation of CaM kinase II to elicit a potentiation or favoring calcineurin to elicit a depression. We will examine Ca2+ -based signaling pathways from the glutamate receptors on synaptic spines to the phosphorylation of the transcription factor CREB in the nucleus with Dr. Tsien. Transgenic animals with reporter genes driven by promoters for regulatory elements for CREB and other transcription factors will be generated to examine transcription at different stimulus frequencies. Single cell PCR will be used to correlate synaptic plasticity and induciton of genes in single cells examined morphologically under the microscopy. Finally, we have developed a method termed indexing which will be optimized to a single cell level and will allow us to compare cDNA from control, LTD or LTP neurons and thereby clone novel plasticity genes.